Studies with novel mutant mouse strains that lack or overexpress M3 mAChRs in hepatocytes only It is well known that an increase in the rate of hepatic glucose production is the major contributor to fasting hyperglycemia in T2D. Interestingly, a brain-liver circuit has been described recently that is thought to be critical for maintaining normal glucose homeostasis. In this circuit, increased insulin and fatty acid levels are sensed in the mediobasal hypothalamus, triggering an increase in vagal outflow to the liver. This increased activity of efferent hepatic vagal nerves results in decreased hepatic glucose production including reduced gluconeogenesis, leading to a lowering of blood glucose levels. Since ACh is the major neurotransmitter of the vagus nerve and exerts its parasympathetic actions via activation of mAChRs, we examined the potential metabolic relevance of hepatocyte mAChRs. We initially demonstrated that the M3 mAChR is the only mAChR subtype expressed by mouse liver/hepatocytes. To assess the physiological role of this receptor subtype in regulating hepatic glucose fluxes and glucose homeostasis in vivo, we employed Cre/loxP technology to generate mutant mice lacking M3 receptors in hepatocytes only. Moreover, to study the metabolic effects of enhanced signaling through liver M3 mAChRs, we also generated mutant mice selectively overexpressing this receptor subtype in hepatocytes. We found that the absence or overexpression of hepatocyte M3 mAChRs had little or no effect on hepatic glucose fluxes and glucose homeostasis in vivo, suggesting that the pronounced metabolic effects mediated by activation of the efferent vagal nerves innervating the liver are mediated by signaling pathways that do not involve the activation of hepatic mAChRs. Studies with novel mutant mouse strains that allow the selective activation of specific beta cell G protein signaling pathways in vivo Impaired functioning of pancreatic beta cells is a key hallmark of T2D. Beta cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific beta cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that beta cell GPCRs are also expressed by many other tissues. To circumvent these difficulties, we developed a novel chemical-genetic approach that allows for the conditional and selective activation of specific beta cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in beta cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (Gq/11 versus Gs). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either beta cell Gq/11 or Gs G proteins. We found report that conditional and selective activation of beta cell Gq/11 signaling in vivo leads to striking increases in both first- and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated beta cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of beta cell Gs triggered qualitatively similar in vivo metabolic effects. Thus, this newly developed chemical-genetic strategy represents a powerful approach to study G protein regulation of beta cell function in vivo. RGS4 is a potent negative regulator of M3 mAChR signaling in beta cells Recent studies suggest that agents that are able to stimulate beta cell M3 mAChR signaling may prove beneficial for the treatment of T2D (Gautam et al., Cell Metab. 3, 449-461, 2006). However, the potential clinical use of direct M3 receptor agonist as antidiabetic drugs may be complicated by unwanted side effects, since M3 mAChRs also play a key role in mediating several other peripheral actions of ACh. We therefore initiated work to identify proteins that are able to modulate signaling through beta cell M3 mAChRs and, hopefully, are endowed with a more favorable pattern of expression. For these studies, we used MIN6 insulinoma cells as a model system. MIN6 cells almost exclusively express the M3 mAChR subtype and treatment of these cells with muscarinic agonists leads to robust increases in intracellular calcium levels and glucose-stimulated insulin secretion (GSIS). Agonist activation of the M3 mAChR triggers a number of cellular events, such as receptor phosphorylation by various kinases, that serve to dampen M3 mAChR signaling (van Koppen and Kaiser, Pharmacol. Ther. 98, 197-220, 2003). Moreover, the lifetime of the receptor-activated G proteins is greatly reduced by the action of RGS proteins, which catalyze the hydrolysis of GTP that is bound to activated G&#945;subunits (note that the RGS protein family consists of more than 30 different members in mammals). We demonstrated that RGS4 mRNA was by far the most abundant RGS transcript detectable in MIN6 cells. RGS4 mRNA was also found to be highly expressed in mouse pancreatic islets. Interestingly, siRNA-mediated knockdown of RGS4 expression in MIN6 cells led to significant increases in muscarinic agonist-induced elevations in intracellular calcium levels and GSIS. We obtained similar results using pancreatic islets prepared from RGS4-deficient mice and control littermates, indicating that RGS4 represents a potent negative regulator of M3 mAChR function in beta cells. We also noted that RGS4 deficiency had little or no effect on the ability of other ligands acting on different beta cell Gq/11- or Gs-coupled GPCRs to enhance GSIS in MIN6 cells or pancreatic islets, suggesting that RGS4 selectively interferes with M3 mAChR function in insulin-containing cells. Several studies have shown that RGS proteins can directly interact with specific GPCRs, most likely within the context of GPCR/RGS signaling complexes containing additional signaling or scaffolding proteins. We therefore speculate that the ability of RGS4 to selectivity regulate M3 mAChRs signaling in beta cells may be due to the existence of M3 receptor/RGS4 signaling complexes. Consistent with this notion, we were able to co-immunoprecipitate M3 mAChRs in a complex with RGS4 in co-transfected mammalian cells. To study the potential physiological role of RGS4 in regulating beta cell function in vivo, we generated mutant mice that lacked RGS4 in pancreatic beta cells only (beta-RGS4 KO mice). Since the beta-RGS4 KO mutant mice did not show any obvious metabolic phenotype under basal conditions, we inject WT and mutant mice showed with bethanechol (2 ug/g, s.c.), a peripherally acting muscarinic agonist. In WT mice, bethanechol induced a transient increase in serum insulin levels, associated with a moderate decrease in blood glucose levels. These effects were absent in beta-M3 mAChR KO mice, indicating that they were mediated by beta cell M3 mAChRs. Strikingly, in the beta-RGS4 KO mice, bethanechol-induced insulin secretion remained very high throughout the entire 1 hr observation period, associated with more robust decreases in blood glucose levels (compared to bethanechol-injected WT littermates). In summary, these observations indicate that RGS4 represents as a potent negative regulator of M3 mAChR-mediated insulin secretion in vitro and in vivo, suggesting that peripherally acting RGS4 inhibitors may prove useful for the treatment of T2D by enhancing signaling through beta cell M3 mAChRs.